The objectives of this proposal are to understand the subunit interactions and conformational transitions of well studied enzymes, yeast hexokinase, liver glucokinase, and aspartokinase-homoserine dehydrogenase of E. coli, in order to interrelate their molecular properties and physiological functioning. The association-dissociation of yeast hexokinase and its hysteretic transition will be studied by ultracentrifugation, spectroscopic, and thermodynamic methods. The changes in regulatory parameters of hexokinase with growth condition and in regulatory mutants will be examined by electrophoretic, immunochemical, and chemical structure in order to determine the reason(s) for the previously observed differences in negative cooperativity, activation by citrate and other effectors, and the slow transition. Glucokinase from rat and pig liver will be isolated and the structural basis for its regulatory properties and participation in a futile cycle with glucose-6-phosphatase determined. The primary structure of liver glucokinase will be compared to that of yeast hexokinase. The previously proposed regulatory mechanism for aspartokinase-homoserine dehydrogenase which involves two sets of four binding sites for the inhibitor, threonine, and two hysteretic transitions will be tested by use of the alternate inhibitor, serine, and by thermodynamic characterization of the molecular transition that occurs on a rapid time scale. These studies will provide the ground work for understanding the role of subunit interactions and hysteretic responses in the molecular and cellular properties of enzymes and lead to a better understanding of the metabolic dysfunction in diabetes and cancer.